U.S. Pat. No. 3,992,522 teaches temperature-sensitive reassortant mutant viruses, method for their production and vaccines derived therefrom. One could not predict from this art, however, that a reassortant virus formed from a human influenza virus and an equine influenza virus would provide a reassortant virus which could be used to provide a vaccine which on the one hand would not cause serious disease in an equine, yet on the other hand would induce effective resistance in an equine to equine influenzal disease produced by challenge with a virulent wild-type equine influenza virus. Temperature-sensitive (ts) mutation affords the possibility of site-specific attenuation for the lower respiratory tract. The acquisition of temperature-mutant defects in a strain of influenza virus has been shown in mice to be associated with diminished virulence, though the antibody producing stimulus remained (British Medical Journal, 1969, 3:757-758). Replication of mutants with markedly restricted growth at 37.degree. C.-38.degree. C. appears to be greatly limited in the lower respiratory tract, the major site of significant pathology, which has a temperature of 37.degree. C. in humans. However, the mutants should grow with reasonable efficiency in the cooler passages of the upper respiratory tract, which have a temperature of 32.degree.-34.degree. C. In this manner, ts mutants grow primarily in the upper respiratory tract and stimulate immunologic defense mechanisms without producing symptoms in the lower tract. Additionally, ts mutants are often partially defective at permissive temperatures (32.degree.-34.degree. C.), and this property offers the possibility of attenuation for the upper respiratory tract as well.
Much of the prior art relative to the present invention is of the literature variety, e.g.:
Murphy et al, "Temperature-Sensitive Mutants of Influenza Virus II Attenuation of ts Recombinants for Man", The Journal of Infectious Diseases, Vol. 126, No. 2, August 1972, pages 170-178.
Murphy et al, "Temperature Sensitive Mutants of Influenza Virus III Further Characterization of the ts-1[E] Influenza A Recombinant (H3N2) Virus in Man", J. of Infectious Diseases, 128:478-487, 1973.
Beare et al, "Recombinant Influenza-A Viruses as Live Vaccines for Man", Lancet, 2:1271-1273, 1971. This Beare journal article does refer, as the title indicates, to a recombinant influenza-A virus as live vaccines for man; however, at page 1272, Table 2, it shows that there is no correlation between restricted growth at 39.degree. and attenuation. This is brought out by the notation that the virulent parent virus (939) is restricted to 39.degree. and the attenuated virus (PR8) replicates well at 39.degree.. Additionally, clone 7 grows well at high temperature and is virulent, whereas clone 64C similarly grows well at 39.degree. but is attenuated. Beare demonstrates simply that the mating of an avirulent virus (PR8) and a virulent virus (939) can give rise to clones of viruses with a spectrum of virulence for man. However, in this article there is no specific characteristic; i.e., temperature sensitivity, associated with this attenuation. The Beare article also utilizes an avirulent virus produced by serial passage in animal and tisue culture rather than by chemical mutagenesis.
Maassab et al, "Hybrid Formation of Influenza Virus at 25.degree. C.", Fed. Proc., 30:413, 1971 [abstract]. This immunology abstract and the parent article, Proc. Soc. Exp. Biol. and Med., 139:768, March 1972, relate to the procedure of Dr. Maassab to produce attenuated viruses of influenza A virus, which is similar to but distinct from the present procedure. Maassab produces an attenuated parent virus by growth of virus at low temperature, a well-known technique for the production of live attenuated virus vaccine strains. Live measles vaccine have been produced in thiss manner. The present chemical technique involves the chemical mutagenesis of influenza A virus and the subsequent isolation of viruses that are temperature sensitive. The end result is to produce viruses that grow at 34.degree. but not at 39.degree., and these are the ts attenuated viruses. The Maassab technique, which involves a transfer of a defect from an attenuated strain to a new wild-type virus by genetic recombination, is similar to the present invention, but the method of production of attenuated viruses is different.
MacKenzie, "Virulence of Temperature-Sensitive Mutants of Influenza Virus", Br. Med. J., 3:757-758, 1969. With reference to this MacKenzie article, it is noted that distinct from the present development, MacKenzie did not work with recombinant ts viruses, although he did demonstrate the ts mutants of influenza A viruses produced by chemical mutagenesis were attenuated for animals. More important, the virus preparations in the journal article were not suitable for human use and no attempt was made to pass these ts defects to different influenza A viruses by recombination.
Recent epidemiological studies (Ingram et al, Equine Infectious Diseases IV (Proc. of the 4th Internat. Conf. Equine Infec. Dis.) Barnes et al eds. Vet Pub. Inc. Princeton, N.J. (1978) p.p. 329-338. Kemen, Proc. Am. Assoc. Equine Practitioners, 20:119-126, 1974; and Kemen, Hoofbeats, January 1976, pp. 62-63), undertaken in areas of high equine density such as racetracks, training areas, shows and breeding farms have shown that the most important agent causing clinical respiratory disease in the horse is the equine influenza virus. Presently available inactivated equine influenza vaccines have several limitations. First, because of occasional deleterious side-effects in vaccinated horses and incomplete protection, many horsemen are abstaining from a regular vaccination program for their animals. Second, these vaccines provide only a short-lived program which lasts only 3-4 months under intense challenge and requires several vaccinations each year to provide adequate protection (Ingram et al, supra; Burrows, Amer. Assoc. Equine Pract., 1979, p. 37-48).
Unlike the human influenza viruses, there has not been significant antigenic drift in the two equine influenza viruses (Burrows, supra). In addition, infection of horses with the wild-type virus provides a high degree of protection as indicated by the high frequency of disease in two- and three-year-olds with little clinical evidence of reinfection in previously exposed older horses (Ingram et al, supra; Kemen, supra). These factors support the idea that the development of an effective live virus vaccine for equine influenza would be valuable in the control of this disease since a live vaccine might stimulate immunity like that of natural infection.
In recent years, conditional-lethal, temperature-sensitive mutants of human influenza A viruses have been produced characterized and evaluated as candidate live virus influenza vaccines in humans with some promising results (Chanock and Murphy, Rev. Infect. Dis., 2:421-432, 1980). The use of ts mutants as vaccines offers several advantages. The location of the ts lesion on the viral genome can be determined and the level of in vitro temperature sensitivity of plaque formation measured in the laboratory (Murphy et al, J. Infect. Dis., 130:144-149, 1974; Spring et al, Virology, 66:542-550, 1975; and Murphy et al, Infect. Immun., 20:665-670, 1978).
Attenuated ts vaccine viruses can be evaluated for genetic stability during production, experimental trials, and later usage in the field with the ts lesion serving as a marker for vaccine virus.
Ts reassortant clones which are restricted in replication in vitro at 37.degree.-38.degree. C. multiply efficiently in the upper respiratory tract of man and hamsters and induce local and systemic immune responses which protect against wild-type influenza virus (Murphy et al, Infect. Dis. 126:170-178, 1972; Murphy et al, J. Infect. Dis., 128:479-487, 1973; Murphy et al, Virology, 88:244-251, 1978; Jennings et al, Fed. Proc., 37:2072-2073, 1978; Mills et al, J. Infect. Dis., 123:145-157, 1971; and Richman et al, J. Infect. Dis., 134:585-594, 1976). However, because the replication of the virus is sensitive to temperature, these viruses replicate inefficiently in the warmer, lower respiratory tract (approximately 38.degree. C. in the horse) and, therefore, should not produce the cough characteristic of the wild-type equine influenza virus infection. It is hoped that the increased stimulation of local and systemic immune systems by viral replication could decrease the need for frequent vaccinations, with perhaps annual vaccination in horses being sufficient (Chanock et al, Viral Immunology and Immunopathology, 1975, Academic Press, NY., P 291-316).